Probiotic delivery system

ABSTRACT

The present invention relates to a probiotic delivery system that is preferably added to a food product. In particular, the invention shows that compacted pellets having a volume of at least 0.02 cm 3 , that comprise, besides viable micro-organisms, arbitrary or eligible components, such as fillers, binder, plasticizer, other functional ingredients and a coating may be added to semi-moist, moist or semi-dry products. The micro-organisms remain viable for a longer time than commercially obtainable preparations of probiotics.

The present invention relates to pellets comprising viablemicro-organisms and a coating, to a delivery system of probiotics, tothe use of the pellets as a delivery system of probiotics in a foodproduct and to a process for obtaining a delivery system to supplementfood products with. The invention further relates to a food productcomprising the pellets.

THE BACKGROUND ART

Probiotic micro-organisms (hereinafter: probiotics) are livingmicro-organisms, which upon ingestion in certain numbers, exert healthbenefits beyond basic nutrition. The beneficial effects that probioticsmay induce are numerous and form part of the knowledge of the skilledperson. As few examples one may mention the reduction of lactoseintolerance, the inhibition of pathogenic bacteria and parasites, thereduction of diarrhoea, activity against Helicobacter pylori, theprevention of colon cancer, the improvement or prevention ofconstipation, the in situ production of vitamins, the modulation ofblood lipids, and the modulation of host immune functions.

Above described beneficial effects are generally valid for very specificstrains of micro-organisms colonising the intestines of mammals, forexamples companion animals, such as pets, and also for humans.

Therefore, there is considerable interest in including probiotics intofoodstuffs. For example, many fermented milk products, such as yoghurts,which contain probiotics are commercially available.

Similarly, for animals, there has been interest in including probioticsinto animal feed. This holds true at least for feed aimed at livestockas well as for pet food.

Many probiotics exhibit their beneficial effect mainly when they arealive. Hence, if they are added to a food product, probiotics are meantto survive the shelf life of the food, and even more, upon consumptionof the food, the passage through the gastro-intestinal tract as far asthe place of colonization.

Therefore, the state of the art is concerned with the problem ofsupplying probiotics together with foodstuff and/or pet food andproviding a prolonged lifetime of the added probiotics. In particular,probiotics are very sensitive to temperature and moisture as in a moistor semi-dry food, while they are comparatively stable in a dryenvironment, for example, characterized by a water activity (a_(w))below 0.2. The prior art is, therefore, concerned with the conservationof living biomass in a humid, moist or even liquid environment atambient and higher temperatures.

EP 0 862 863 provides a ready-to-eat cereal product comprising agelatinized starch matrix including a coating or a filling whichcomprises a probiotic. Accordingly, spray-dried probiotics are mixedinto a carrier substrate, which may be water, fat or a protein digest,and the mixture is then essentially sprayed onto the cereal product. Ofcourse, the cereal product must itself have low water activity to ensurea prolonged lifetime of the probiotics.

EP 0 704 16 is concerned more concretely with the preservation of lacticacid bacteria in moisture food. The spray-dried bacteria are added to acomposition comprising fats, fermented milk powder and saccharides. Thiscomposition is then indended as the filling of a confectionary product.This invention avoids the detrimental effects of water by embedding theprobiotics in a matrix rich in fat or oil and therefore risks to shift abalanced nutritional composition of a food product to the negative.

EP 0 180 743 discloses micro-organisms that are suspended in an oilphase and are encased by at least one protective layer, which iswater-soluble (water-soluble derivatives of cellulose or starch, gums orpectins).

A good stability of micro-organisms in a micro-encapsulated form iscommercially available from Cerbios-Pharma SA, Bioferment, Lugano,Switzerland under the product name of LBC ME 10, for example. Amicroscopic view on a cut of the small capsules (diameter about 700 μm)shows that probiotics attached to a carrier are coated by several thinlayers that protect the probiotics. These products are characterised bya relatively high stability also in moisture environments, but are alsoexpensive to produce, since several layers must be added, to avoid waterentering the micro-capsules.

Therefore, a need exists for a delivery system of probiotics, whichprovides, with respect to the existing prior art, a still prolonged lifespan of the probiotics in a liquid, moist or semi-moist environment.

It is, in particular, the challenge of the present invention to providestable probiotics or a probiotic delivery system, added to a foodproduct that has itself an a_(w) value above the optimal value forprobiotics to survive. Furthermore, the probiotics should preferably beprovided in a form, which does not substantially differ from ordeteriorate (from a nutritional and organoleptic point of view) the foodproduct to which they are added.

SUMMARY OF THE INVENTION

Remarkably, it was found that by compacting dried micro-organismstogether with a matrix, which may consist of dried food material, and bycoating the pellets with a food-grade moisture barrier an excellentstability over storage time is obtained.

Consequently, in a first aspect, the present invention provides a pelletcomprising a compacted inner matrix and at least one coating, whereinthe inner matrix comprises viable micro-organisms and the coatingcomprises a moisture barrier, characterised in that the pellet has avolume of at least 0.02 cm³.

In a second aspect, the present invention provides pellets within a foodproduct, wherein the pellets are the pellets according to the inventionand the inner matrix of the pellets comprises at least one component ofthe food product.

In a third aspect, the present invention provides a delivery system ofprobiotics, which comprises the pellets according to the invention.

In a fourth aspect, the present invention provides the use of thepellets according to the invention as a delivery system for probioticsin a moist, semi-moist, or semi-dry food product.

In a fifth aspect, the present invention provides a process forobtaining a pellet to supplement a food product with viablemicro-organisms, which comprises the steps of mixing a preparation ofmicro-organisms and further components, drying the mixture to an a_(w)below 0.3, compacting the mixture under pressure to obtain pelletscomprising a volume of at least 0.02 cm³, and coating the pellets with amoisture barrier.

In a sixth aspect, the present invention provides a food productcomprising the pellets according to the invention, wherein the foodproduct and the inner matrix of the pellet share at least one component,ingredient or constituent.

In a last aspect, the present invention provides a particulate food,food additive, supplement or pharmaceutical product consisting of amixture of at least two types of particles, one being the probioticpellet according to the invention.

An advantage of the present invention is that it provides a significantimprovement of the stability of probiotic micro-organisms applied insemi-dry and/or humid particulate foodstuffs.

Another advantage of the present invention is that the processing iseasy and straightforward.

Yet another advantage of the present invention is that it provides asuitable delivery vehicle for further functional ingredients, inparticular also prebiotic fibres, which in turn may improve thephysico-chemical characteristics of the pellet according to theinvention.

In the figures,

FIG. 1 schematically illustrates an example of the pellet (1) accordingto the invention. The pellet (1) comprises an inner matrix (2), whichcomprises probiotics (3). The pellet further comprises a moisturebarrier (4). The FIGS. 1 a and 1 b distinguish different embodiments,wherein the probiotics are homogeneously dispersed (FIG. 1 a) oraccumulated in the centre (FIG. 1 b). It should be noted that the shapeand the ratios between thickness of coating, inner matrix and probioticpreparation is arbitrary and only serves the purpose of illustration.

FIG. 2 shows the storage stability (recovery in % of cfu/g versusstorage time) of Enterococcus faecium stored at 30° C. and 70% RH(relative humidity). The figure distinguishes (●) values obtained withthe commercially available micro-capsules obtained from Cerbios-Pharma,Lugano, Switzerland under the designation SF68 and the coated pelletsaccording to the invention (♦).

DETAILED DESCRIPTION OF THE INVENTION

Within the context of this specification the word “comprises” is takento mean “comprises, among other things”. It is not intended to beconstrued as “consists only of”.

Within the context of the present invention, the term “food product” isintended to encompass any consumable matter. Hence, it may be a productintended for the consumption by humans, but the term also encompassesproducts to be consumed by animals, for example pets, such as dogs,cats, rabbits, guinea pigs, mice, rats, birds (for example parrots),reptiles and fish (for example goldfish). However, the term alsoincludes food to be consumed other domesticated animals, for examplefeed products for livestock, for example, cattle, horses, pigs, sheep,goats, buffaloes, camels, and the like.

The term “pellet”, in the context of the present invention, is notintended to refer to any specific form. In the contrary, a “pellet”, inthe sense of the present invention, may assume any form obtainable bycompaction. For example, a pellet may have the form of a sphere, cube,pyramid, tablet or any classic, modified or complex three-dimensionalform. Furthermore, fancier forms may be conceived. For example, if thepellets are intended as a probiotic delivery system for pet-food, theymay have the form of bones, rods, rings, the form of animals, forexample mice, or other objects. The compaction techniques of today allowthe preparation of almost any three-dimensional structure.

In the context of the present invention, the term “probiotic” isintended to refer to any micro-organism that is wished to be consumedowing to any beneficial effect it may have on its consumer.

In the context of the present invention, the term “moisture barrier”refers to any substance that may be used to coat the pellets as defmedabove and is useful to slow down the water absorption by the pellets tobe coated.

In the context of the present invention the following a_(w) values fordefining “moisture” are used as practical approach: moist denotes ana_(w) of 0.7 or above, semi-moist an a_(w) between 0.5 and 0.7 andsemi-dry and a_(w) between 0.3 and 0.5.

In the context of the present invention, the word “matrix” in theexpression “inner matrix” is not intended to be restricted to anyspecific component or selection of components. Of course, it usuallycomprises food-grade ingredients, the consumption of which isnutritionally safe. However, one of the advantages of the presentinvention is the very flexibility the skilled person is given withrespect to the matrix. The matrix may serve as a carrier for theprobiotics, optionally together with a binder.

The word “binder”, in the context of the present invention, may refer toany food-grade substance that has the property of giving other foodingredients the ability to be compacted. It is generally understood thatbinders have the ability to be plastically deformed during compaction,thereby delivering a sufficiently durable tablet structure. In addition,binders may have adhesive, gluey or sticky properties.

The word “plasticizer”, in the context of the present invention, mayrefer to any foodgrade substance that has the property of softening oneor more of the other ingredients used in preparing the compacted pellet.Preferably, plasticizers are used that have the capacity of bedewing orwetting the surface of other components of the inner matrix and by thisway support the compaction of the inner matrix even at a low wateractivity.

The word “functional food”, in the context of the present invention,refers to any food product, or any food ingredient that provides to theconsumer any benefit that goes beyond basic nutrition. Probiotics (seeabove) that may have beneficial effects on the composition and metabolicactivity of the gut microflora of the host and on its immune system, mayserve as an example. Likewise, prebiotics, fiber, vitamins,anti-oxidants, psycho-stimulating molecules, such as caffeine, may serveas a purely arbitrary, exemplary but illustrative choice of possible“functional food” ingredients.

The term “further components”, in the context of the further componentsof the inner matrix, refers to all other components than bacteria_(w) Incase that the bacteria are attached to a specific carrier, this carriermaterial is likewise not part of the “further components”.

All lists of ingredients or parts of the pellet or probiotic deliverysystem given within this description are regarded as non-exhaustivelists. It will always be possible, though not mandatory, to select anycombination of the ingredients or substances of one or all of theselists.

Percentages are given in percent by weight, unless otherwise indicated.Preferably, in an embodiment the pellet according to the presentinvention has a volume in the range of 0.01 to 100 cm³, preferably 0.02to 50 cm³, more preferably 0.125 to 30 cm³ and most preferably 0.3 to 8cm³. For example, the volume may be in the range of 0.4 to 6 cm³, 0.5 to3 cm³, or of 0.6 to 2.25 cm³.

In another embodiment according to the present invention, the innermatrix of the pellet according to the present invention before orshortly after the coating, is characterised by a water activity of below0.3. Preferably, the water activity is below 0.2, more preferably about,equal to or below 0.1. For example, the water activity is in the rangeof 0.01 to 0.09.

In a further embodiment, the inner matrix of the pellet according to thepresent invention has an envelope density of more than 0.8 g/cm³.Preferably, it has an envelope density of above 1.1 g/cm³, morepreferably above 1.3 g/cm³.

The envelope density is a measurement that indicates the specific weightof an object including pore spaces up to the plane of the surface. Thisquantity is specifically suitable for tablets or compacted entities, andmay be assessed with a Micromeritics® Geo Pyc 1360 apparatus, forexample.

Another way of expressing specific weight of compacted entities, forexample is the absolute density. The absolute density of the uncoatedpellet is preferably above 1 g/cm³, more preferably above 1.2 g/cm³ andmost preferably above 1.5 g/cm³. The absolute density may be assessedwith a Micromeritics® Accu Pyc 1330 apparatus, for example.

Definitions of envelope and absolute densities are known to the skilledperson but may be derived, for example from Webb, Pa.; Orr, C.Analytical methods in fine particle technology. Micromeritics InstrumentCorporation, Norcross, Ga., 1997.

Generally, it may be said that the envelope density is the overalldensity of a body as determined from its volume including closed andopen pores up to the plane of the surface. The absolute density is thedensity of a body as determined from its volume including closed pores(but excluding the open pores). The matrix or real density (notindicated in the embodiments above) is the density of a body asdetermined by the volume of the matrix excluding both open and closedpores.

Generally, the envelope density of the uncoated pellets is between about50% and 100%, preferably between 70% and 100% and more preferablybetween 80% and 100% of the absolute density of the uncoated anduncompacted components of the inner matrix.

In still another embodiment, the pellet according to the presentinvention comprises 10⁵ to 10¹² viable micro-organisms (cfu).Preferably, it comprises 10⁵ to 10¹¹ cfu, more preferably 10⁶ to 10¹⁰cfu. For example, the pellet according to the invention may comprise 10⁶to 10⁸ cfu.

The above values are dependent on the size of the pellet and the numberof cfu added. The recommended values of cfu may also be expressed in gof pellet, disregarding the size of the pellet. Hence, the pelletaccording to the invention preferably comprises 10⁵ to 10⁹, morepreferably 10⁶ to 10⁸ cfu/g.

In still a further embodiment, the inner matrix of the pellet accordingto the present invention further comprises ingredients selected from thegroup of digestible starches, resistant starches, other fibre, milledcereals, dried and milled vegetables, cellulose and cellulosederivatives, pet food, maltodextrin, chicory flour, protein isolates,yeast extracts and mixtures thereof.

In yet a further embodiment, the coating of the pellet according to theinvention comprises a food-grade moisture barrier.

In an embodiment of the process according to the invention, the furthercomponents comprise at least part of the ingredients of the foodproduct.

Without wishing to be bound by theory it is postulated that by addingdried probiotics to food material or specific food grade ingredients,drying, compacting them to relatively large particles or pellets (≧0.02cm³), and coating them with a material that serves as a moisturebarrier, a high stability of the encapsulated probiotics is reached.This may be so in part because the ratio of volume and surface is muchbetter exploited than in encapsulated or dried probiotics so far known.The compaction and the moisture barrier flurther support the optimisedratio and allow a storage stability of probiotics in a moistureenvironment that was so far not achieved.

In order to prepare the pellets according to the present invention, asingle or a mixture of different possible micro-organisms, the furthercomponents of the inner matrix and the hydrophobic substance may beselected.

As a micro-organism, any micro-organism may be selected. Preferably, amicro-organism exerting beneficial effects on health and welfare onhumans or animals, such as pets, for example cats or dogs, lifestockanimals, for example, pigs, cattle, buffaloes, sheep or goats isselected. Preferably, the micro-organism is a probiotic micro-organism.

The literature mentions some of the micro-organisms that may be used tocarry out the present invention, for example in EP 0 862 863A2, inparticular on page 3.

Examples of suitable probiotic micro-organisms include yeasts such asSaccharomyces, Debaromyces, Candidaw Pichia and Torulopsis, moulds suchas Aspergillus, Rhizopus, Mucor, and Penicillium and Torulopsis andbacteria such as the genera Bifidobacterium, Bacteroides, Clostridium,Fusobacterium, Melissococcus, Propionibacterium, Streptococcus,Enterococcus, Lactococcus, Kocuriaw, Staphylococcus, Peptostrepococcus,Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus,Oenococcus and Lactobacillus. Specific examples of suitable probioticmicro-organisms are: Aspergillus niger, A. oryzae, Bacillus coagulans,B. lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis,B. natto, Bacteroides amylophilus, Bac. capillosus, Bac. ruminocola,Bac. suis, Bifidobacterium adolescentis, B. animalis, B. breve, B.bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, B.thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcuscremoris, E. diacetylactis, E faecium, E. intermedius, E. lactis, E.muntdi, E. thermophilus, Escherichia coli, Kluyveromyces fragilis,Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus,L. brevis, L. case4 L. curvatus, L. cellobiosus, L. delbrueckii ss.bulgaricus, L farciminis, L. fermentum, L. gasseri, L. helveticus, L.lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei,L. salivarius, Leuconostoc mesenteroides, P. cereviseae (damnosus),Pediococcus acidilactici, P. pentosaceus, Propionibacteriumfreudenreichii, Prop. shermanii, Saccharomyces cereviseae,Staphylococcus carnosus, Staph. xylosus, Streptococcus infantarius,Strep. salivarius ss. thermophilus, Strep. thermophilus, Strep. lactis.

For example, a probiotic strain or strains may be selected from thegroup comprising Bacillus licheniformis (DSM 5749), B. subtilis (DSM5750), Bifidobacterium lactis (DSM20215), strains of Enterococcusfaecium (e.g. NCIMB 10415; NCIMB 11181; NCIMB 30098; DSM 3520; DSM 4788;DSM 4789; DSM 5464; DSM 7134; CECT 4515), E. mundtii (CNCM MA 27/4E),strains of Saccharomyces cereviseae (e.g. BCCM/MUCL 39885; CBS 493 94;CNCM I-1077; CNCM I-1079; NCYC Sc47), Lactobacillus casei (NCIMB 30096),L. farciminis (CNCM MA 67/4 R), L. johnsonii (I-1225 CNCM),Lactobacillus paracasei (I-2116 CNCM), L. plantarum (CNCM I-840), L.rhamnosus (DSM 7133), P. acidilactici (CNCM MA 18/5 M), Streptococcusinfantarius (CNCM I-841), Streptococcus thermophilus (TH4, Chr. Hansen,DK), and mixtures thereof, for example.

Further examples of probiotic species with exemplary, deposited strainsof the species according to the present invention may be selected fromthe group comprising Lactobacillus reuteri (CNCM I-2452, CNCM I-2448,CNCM I-2450, CNCM I-2451), Lactobacillus rhamnosus (CNCM I-2449),Lactobacillus acidophilus (CNCM I-2453), and mixtures thereof. Thestrains mentioned in this paragraph may be particularly suitable forpets.

The micro-organisms are preferably in a dried form, or for example in aspore form for micro-organisms which form spores. The drying ofmicro-organisms after production by fermentation is known to the skilledperson. For example, EP 0 818 529 (SOCIETE DES PRODUITS NESTLE), where adrying process of pulverisation is described, or WO 0144440 (INRA).Usually, bacterial micro-organisms are concentrated from a medium anddried by spray drying, fluidised bed drying, lyophilisation (freezedrying) or another adequate drying process. For example, micro-organismsare mixed with a carrier material such as a carbohydrate, for examplesucrose, lactose or maltodextrin, a lipid or a protein, for example milkpowder during or before the drying. If a carrier material is used, itmay also form part of the inner matrix.

However, the micro-organisms need not necessarily be present in a driedform as from the beginning. It may also be conceived to mix themdirectly after fermentation with the frther components of the innermatrix (see below) and to perform a drying process thereafter. Such anapproach could be deducted from WO 02/065840 (SOCIETE DES PRODUITSNESTLE).

In a preferred embodiment, the micro-organisms are in the form ofparticles with a particle size of at least 100 μm, preferably at least200 μm, more preferably at least 300 μm. For example, the particle sizemay be about 1 mm. These numbers refer to the average diameter of theparticle.

Preferably, the particles comprise significant amounts of inertamorphous carbohydrates, in which the micro-organisms are embedded.Preferably, the particles comprise, in percent by weight of total drymatter, 10-90%, preferably 30-80%, more preferably 40-70% of inertcarbohydrates. Examples of inert carbohydrates are maltodextrins,starches, low molecular weight sugars (sucrose, lactose, maltose,mannitol, and the like) and hydrocolloids (pectin, guar, xanthan, gumacacia).

The particles comprising micro-organisms and inert carbohydrates arepreferred, because the micro-organisms are less susceptible tosubsequent compaction and thus better survival obtained.

Suitable particles are obtained by mixing the micro-organisms with theinert carbohydrates after fermentation and spray drying or fluidized-beddrying the mixture according to established procedures known to personsskilled in the art. State of the art techniques for spray drying andfudized-bed drying are described in for instance K. Masters, SprayDrying Handbook, 5^(th) ed. Longman, Harlow (1991) and in K. Dewettinckand A. Huyghebaert, Fluidized bed coating in food technology. TrendsFood Sci. Technol. 10, 163-168 (1999) and references contained therein.

Furthermore, the further components of the inner matrix may be selected.One of the advantages of the present invention is the high flexibilityand variety with respect to the further components of the inner matrix.This high variety is reflected by the lists of possible molecules orfunctions that may be added to the inner matrix.

Therefore, the further components may be regarded as “fill-up” or“filler” components and their choice is almost arbitrary and freelyeligible. In case that the micro-organism is already mixed with furtheringredients, for example carrier materials or protective agents, it iseven possible that no further component must be selected.

Generally, if further components of the inner matrix are selected,substances with a high hygrocapacity are generally advantageous withrespect to the ability of the coated pellet to retard the increase ofthe internal water activity. Even if water is absorbed by the innermatrix of the pellet, the water activity remains relatively low becauseof the high capacity of the inner matrix to absorb moisture. Polymericcarbohydrates, for example, have a high hygrocapacity.

Preferably, the futher components of the inner matrix may be selected ina way that compaction of the inner matrix is possible. Generally, thismay be also achieved by a suitable binder and/or plasticizer.

For the sake of convenience and to avoid confusion in view of the highvariety or choice in the selection of the further components of theinner matrix, these are exemplary discussed as fillers, functionalingredients, lubricants, plasticizers and binders. It is understood thatthese classes do not cover completely different ingredients, but theremay be an even substantial overlap. For example, some plasticizers mayalso serve as binders, or fillers may also comprise functionalingredients, such as fibres, for example.

Fillers, that may help to increase the volume may be selected from thegroup comprising starches; resistant starches, low molecular weightsugars, for example, lactose, dextrose, sucrose, and/or mannitol,microcrystalline cellulose, modified starches, for example, amylodextrinhydrogen octenylbutanedioate; and/or starch n-octenylsuccinate,proteins, for example milk-, pea-, soy-, meat-, poultry-,gluten-protein, hydrocolloids, milled cereals, dried and milledvegetables, animal meal, milk powder, cocoa powder, milled biscuit, ormixtures thereof.

Functional ingredients may be selected to provide further benefit to thepellet or delivery system according to the present invention. They maycomprise, apart from the probiotics, prebiotic fibres, for examplefructo-oligosaccharides (FOS), polyfructoses, for example, inulin orlevan, resistant starches, for example retrograded starch, dextrans,arabinogalactans, for example acacia gum, galactomannans, for exampleguar, galactooligosaccharides, isomalto-oligosaccharide,maltooligosaccharide, maltodextrins, and mixtures thereof, for example.

Fibres may be differentiated in soluble and non-soluble fibre. Examplesof soluble fibers comprise inulin, pectin, 6-glucans (smallcellulose-type branched glucose polymers), gumarabic, tragacanth,mucilages, guar and locust bean gum, agar, carageenans, alginates,xanthan and the like. Most of these soluble fibres are fermentable forthe largest part. Examples of insoluble fiber comprises cellulose (forexample derived from oat hull, soy-beans, cereal bran) and hemicellulose(mostly branched arabino-xylans or galactans, e.g from cereals, potatoesor soybeans). Most of these insoluble fibres are partly fermentable ornon-fermentable.

Functional ingredients may also comprise trace elements, minerals,vitamins, antioxidants, sterols, antioxidants, fatty acids, proteins,for example enzymes, and/or other functional molecules.

Examples of vitamins and/or antioxidants may be selected from the groupcomprising carotenoids, such as lycopene, α-, β-, or γ-carotin,xanthophylls, vitamin A1, vitamin A2, tocopherols, for example vitamineE, vitamine C, and mixtures thereof.

Examples of fatty acids may be selected from the group comprising long-,medium chain saturated or unsaturated, mono-, di-, or triacylglycerols,and mixtures thereof, for example.

Examples of enzymes may be selected from the group comprising proteases,peptidases, lipases, hydrolases, and cocktails thereof, for example.

Other functional molecules may be selected from the group ofbacteriocins, chondroitin sulfate, soy isoflavones, nucleotides,nucleosides, isothiocyanates, cruciferous extracts, for example frombroccoli, sulfloraphane, and mixtures thereof, for example.

Ingredients or molecules that have other functions may be added to theinner matrix. These ingredients or molecules may enhance intestinalfunctions, maintain or enhance skin integrity, prevent skin damage (forexample, UV induced) and stress response, improve coat conditions,prevent infection, maintain or improve oral health, enhance and/ormaintain vision, prevent allergy, modulate immune functions, preventobesity, provide weight control, lower the risk of diabetes and/orartherosklerosis, control triglycerides in blood and tissues, enhancenutrient absorption, improve brain development and enhance and/ormaintain cognitive functions, prevent vascular disease, for exampleheart attack or stroke.

In addition or alternatively, ingredients or molecules may be providedthat maintain and improve of kidney, liver and pancreas functionality,improve joint health, prevent arthritis, improve bone development duringgrowth, improve or enhance maintenance of bone mass during adulthood,increase mineral (for example calcium) and vitamin absorption andutilisation from food, prevent and/or slow down osteoporosis, improvemuscle growth, performance and/or recovery, have anti-inflammatoryproperties, improve breath, enhance immune functions, haveanti-pathogenic activity and/or inhibitory activity.

Binders and/or plasticizers may be added to the components of the innermatrix, if necessary, to improve the compaction properties of thecomponents of the inner matrix. It may be, however, that the furthercomponents and/or the micro-organisms are themselves sufficiently“sticky” or adhesive to allow for compaction. In this case, a specific“binding component” may be omitted. However, if one or several bindersand/or plasticizers are added to support the formation of pellets bycompaction, preferably food-grade ingredients are used.

Examples for plasticizers may be selected from the group comprisingpolyols (for example, glycerol, sorbitol, propyleneglycol), alcohols(for example, ethanol, propanol, butanol, isopropanol, isobutanol,butanediol).

Examples of binders may be selected from the group comprising suitablepolysaccharides, for example starches (native starch, waxy maize starch,hydrolyzed starches, maltodextrins, pregelatinized starches),polyfructoses (chicory flour, inulin), hydrocolloids,polyvinylpyrrolidone, hydroxypropyl methylcellulose, for example.

Lubricants may serve the purpose of facilitating the release of thepellet from the tabletting, pelletting or briquetting mould by reducingthe friction forces between the pellet and the forming mould and, ifrelevant, the tabletting punch. Moreover, they may promote the flow ofthe pellet premix in the hopper and in the tabletting or pellettingmould. Examples of lubricants may be selected from the group comprisingstearic acid, stearic acid salts, stearic acid derivatives, talc,polyethylene glycols, surfactants, and waxes.

Since it is the objective of the present invention to add the pellets toa food product, it is an outstanding advantage that the inner matrix maycomprise one, several or all components of the food product to which thepellets will be added.

For example, if the pellets are to be added to a pet food, the innermatrix may be supplemented, partially or totally, by the pet food towhich the pellets will be added. Hence, pet food may just be milledand/or dried in either a cooked or uncooked state and thus used as part(for example filler) of the inner matrix of the pellets.

Accordingly, if the pellets are added to breakfast-cereals, the innermatrix may comprise cereals. Or, if the pellets are added to a snack aschips, for example, the inner matrix may comprise potato starch or otheringredients, such as flavours, used to prepare the chips. This inventiveconcept may be extended to any food product.

Also the moisture barrier coating the compacted components of the innermatrix may be selected. In principle, any food-grade substance havingwater repelling or impermeable properties may be selected. The skilledperson is usually available to select one or mixtures of suitablemoisture barriers. Nevertheless, a list is given below for illustration,from which at least one or mixtures may be selected.

Hence, suitable moisture barriers may be, for example, waxes .(paraffinwax, beeswax (white and yellow), carnauba wax, candellila wax,microcrystalline wax, rice bran wax, cetyl ester wax, shellac,emulsifying wax, lanolin, hydrogenated castor oil, jojoba oil), fattyacids (for example, oleic acid, stearic acid, palmitic acid, lauricacid) and their salts (for example, sodium, calcium, magnesium,aluminium); fatty acid derivatives (for example, cetyl palmitate,acetic, lactic, citric and tartric mono and di glyceride fatty acids,sodium lauryl sulfate), esters of fatty acids (for example, isopropylpalmitate, isopropylmyristate), monoglycerides, diglycerides andtriglycerides (for example, MCT oil, triglycerides based on coconut/palmkernel oil), derivatives of monoglycerides, diglycerides andtriglycerides (for example, polyglyceric esters of fatty acids,propyleneglycol esters of fatty acids, vegetable oils and fats (forexample, rapeseed, sesame, cornseed, nut, cottonseed, peanut, sunflower,linseed, olive, soy bean, cocoa butter) hydrogenated or hardenedvegetable oils and fats, fractionated vegetable oils and fats, oils andfats of animal origin (for example, beef, poultry, pork, lamb; forexample, beef tallow, lard), hydrogenated or hardened oils and fats ofanimal origin fractionated oils and fats of animal origin, dairy fats(for example, milkfat, fractionated milkfat, butterfat), proteins (forexample, gluten, zein, sodium and calcium caseinate), phospholipids (forexample, lecithin), carbohydrates (for example, cellulose and cellulosederivatives (for example, hydroxypropyl methylcellulose, ethylcellulose,methylcellulose, carboxymethyl cellulose), hydroxypropylated starch,carrageenans), sorbitan esters (for example, mono-oleate, -palmitate,-stearate, trioleate), mineral oils and fats (for example, paraffin),chocolate polyvinylalcohol, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(lactic acid), pharmaceutical glaze,latex, methacrylic acid copolymer, poloxamer, polyoxylethylenederivates, tocopherols, sterols, carotenoids, dimethicone, sucroseesters of fatty acids and sucroglycerides.

In a preferred embodiment, the moisture-barrier is a lipid-basedcoating.

Mixtures and laminates of mentioned ingredients may comprise, forexample, casein—acetylated monoglyceride, stearic acid, beeswax; casein,gelatin, soy protein, zein—fatty acid amylose ester; zein, albumen,casein, gelatin, soy protein—vegetable oil; nitrocellulose—wax;zein/vegetable vax-oil laminates.

The preparation of the pellets after selection of the micro-organism andthe further components of the inner matrix may occur in any suitableway. A recent review of the state of the art in the technology andmaterials science of tablet compression is given in “PharmaceuticalPowder Compaction Technology”, Alderbom G and Nystrom C, eds, MarcelDekker, New York (1996).

A few principle steps of preparation of the pellets may usually comprisethe steps “mixing, drying, compacting and coating”. The sequence ofthese steps may be varied in a way that corresponds to common sense. Forexample, the above-mentioned sequence may be modified to “mixing,compacting, drying and coating”.

As a variation, ingredients of the inner matrix, comprising moistfillers, optionally binders or functional ingredients, besidesprobiotics, may be mixed and compacted, whereby a relatively high watercontent supports the compaction. Thereafter, the compacted pellet may bedried and coated.

Most of these steps, for example “mixing” and “drying”, may besubdivided, for example “mixing only few of the ingredients, dryingthem, adding other ingredients to the mixture, compacting, drying againand coating”.

As the above paragraphs illustrate, the preparation of the pelletsaccording to the present invention is very flexible. It is preferred,however, that shortly before or at the moment of coating the compactedinner matrix, the inner matrix has a relatively low water activity(a_(w)), for example an a_(w) below 0.3, preferably below 0.2, morepreferably below 0.15.

For example, a dry mix of the micro-organisms and the furthercomponents, is prepared by mixing all components. Then the mix may bedried to an a_(w) below 0.3, preferably below 0.2, more preferably below0.15 and most preferably around, equal to, or below 0.1. Possible dryingdevices comprise convection ovens, belt dryers, vacuum dryers, fluidizedbed dryers, rotary dryers, just to mention a few.

The moment of drying in the process of obtaining the pellets is notcrucial. For example, the drying may take place after compaction, ifcompaction is easier with slightly moistened components. However, theabove-indicated a_(w) values are preferably achieved before the coating,to the end that the low a_(w) values are preserved within the coatedpellets.

The number of viable cells (cfu) added to the premix of the inner matrixis dependent on the intended consumption of pellets per day, which inturn is also dependent on the size of the pellet and/or the number ofpellets added to a food product. Further variables are the density oroccurrence the pellets will finally have in the food product, theconcentration of the micro-organisms in a dried form, the serving sizeof the food product, just to mention a few.

The skilled person is instructed to calculate the number of cfu to beadded to the mixture by being aware that the daily dose of probiotics isconsumed.

If one pellet is intended to comprise all probiotics of an entire dailyserving, or if no other meal comprising probiotics is intended to beconsumed than one pellet, the pellet preferably comprises the daily doseof probiotics. In this case, one pellet comprises about 10⁵ to 10¹⁴,preferably 10⁶ to 10¹³, more preferably 10⁷ to 10¹¹ cfu/day.

If, alternatively, the expected daily consumption of pellets comprises 2to 10 pellets/day, the above-indicated ranges of cfu per pellet may bedivided by the corresponding number.

Advantageously, the amount of added micro-organism is calculated in away that an effective amount of micro-organism will be consumed byconsuming one or two, optionally three servings of the respective foodproduct to which the pellets will be added.

If a binder, lubricant and/or plasticizer is used, it may be selectedfrom the lists above. For example, glycerol, in the range of 0.15 to20%, preferably 0.5 to 10%, calculated as the total weight of the innermatrix, may be sprayed onto the surface of inner matrix components andthe probiotic preparation.

Upon the optional addition of a binder, lubricant and/or plasticizer,which may already be added to the premix before the drying step above,the resulting mix may be compacted, for example at a sufficientcompaction pressure. Usually, it may be conceived that very highpressures, for example pressures that are substantially above 10′000 barshould be avoided, because the bacteria may be destroyed. However, the(probiotic) bacteria, depending on their condition (dried, wet, on acarrier) may support varying pressures.

The lower limit of a compaction pressure is dependent on the“compaction”—properties of the inner matrix. In principle, thecompaction pressure may be adjusted following the criterion that areasonable consistency and/or stability of the pellet is obtained bycompaction.

The upper limit is principally unidentified, but if the food product isintended for being chewed, the compaction pressure is preferablyselected in a way that the compacted pellets will not be too hard toavoid damages of teeth.

Besides the fact that the compaction pressure may be selected from abroad range, it was found that pressures in the range of 100 to 10′000bar, preferably 200 to 9000 bar and more preferably 300 to 8000 bar. Forexample, compaction may be performed at a pressure in the range of 600to 8000 bar. The pressures indicated above strongly depend on the statusof the micro-organism. If they are still moist, pressures above 4500 barmay destroy them. However, if the probiotics are in the form of spores,much higher pressures may be applied.

Compaction may be achieved by any suitable compacting device. Examplesare rotary tablet presses, eccentric tablet presses, single and doublepunch tablet presses, single and multi-layer tablet presses, briquettingmills, pellet mills, for example.

The pellets may have the volumes as indicated above and be of anysuitable, adequate or desired form. For example, they may have the formof spheres, cubes, pyramids, tablets or any classic, modified or complexthree-dimensional form. Furthermore they may have a form thatcorresponds to the food product to which the pellets are added. Forexample, if the pellets are added to a pet food for dogs, they may havethe form of bones, animals, cats or other forms that fit well with thefood product.

Depending on the components that accompany the micro-organisms, thegeneral process for compacting the mixture may be freely modified,supplemented and adjusted.

If the a_(w) of the compacted matrix is not yet sufficiently low (seevalues given above) a drying step should be introduced before thefollowing coating step (see above).

Then, the compacted pellets comprising the inner matrix may be coated tofurther protect the micro-organism from deleterious effect of subsequentabsorption of water during the shelf-life of the food product. Thecoating may be done by any suitable coating technique, for example,spraying, melt or solvent coating equipment, fudized bed coater, drumcoater or pan coater, just to mention a few. The pellets are coated withmoisture barrier, which is preferably foodgrade, as already exemplified.

Preferably, the amount of coating may provide from 2 to 30%, preferably5 to 20%, more preferably 8 to 18% of the uncoated pellets.

It is understood for the purpose of the present invention that thementioned coating process can be carried out either in one or inmultiple steps and that the term “moisture barrier” refers to either asingle layer of one compound or a mixture of compounds or to multiplelayers of one or more compounds with said barrier properties.

Due to the coating, the drying of most or all of the components of theinner matrix, and the compaction, a low a_(w) of the pellets may bemaintained for a prolonged time.

Thereafter, the food product the probiotics are intended for may besupplemented with a sufficient amount of the pellets according to theinvention. The food product may have a moisture content significantlyabove the a_(w) of the pellets. For example, if the pellets are added afood product with an a_(w) of ≧0.2, ≧0.4, ≧0.5 or even ≧0.6, asurprisingly high viability over a long storage time is achieved.

The food product may be any food product to which the beneficialfunction of probiotics is wished to be added. For example, it may be apet food, including treats. However, it may be any food, intended forany animal. For example, the food product may be a particulate food orfood ingredient, such as certain semi-dry pet foods, breakfast cereals,breakfast flakes, -crisps, or -puffs, snacks, chips, dips, biscuits,candies, confectionery, chocolate, bars, muesli, instant beverages intablet or pellet form, bouillon cubes, instant soup and sauces in tabletor pellet form, oral cosmetics. Of course, the food product may be afood product that is manufactured with particulate ingredients, such ascertain bars, which consist of compressed particulate ingredients, forexample.

As hinted at above, the number/weight of pellets intended to be added toa food product depends on several factors, amongst which (I) the cfu/gof pellets, (II) the serving size of the food product, and (III) the“effective dose”, that is, the amount of cfu that preferably be consumedto obtain the desired effect. The effective daily dose of manyprobiotics with respect to many animals lies in the range of 10⁷ to10¹⁰, for example between 10⁸ to 10⁹ cfu per day and individual.

For example, the pellets may used exclusively, that is, the pellets formthe entire food product, for example as a treat or a supplement. In theinstance of a particulate pet food, this would mean that all particlesof the food are constituted by the pellet according to the invention.

In another example, the pellet may be added to a food product in anamount of 1 to 70%, preferably 3 to 50%, more preferably 5 to 30% andmost preferably 8 to 20% to a food product. These percentages may be byweight or by number of particles, such as pet food kibbles, for example.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication. It is repeated that the gist of the present inventionresides also in the fact that an unlimited variety of food ingredientsmay be used to form the inner matrix of the pellets. Percentages andparts are by weight unless otherwise indicated.

EXAMPLE 1 Preparation of Pellets for Pet Food

Pellets are prepared by compaction of a powder matrix and are coatedwith a food grade component providing a high moisture barrier. Theentire mixture comprises chicory flour, maltodextrin (DE2-6), andFRISKIES Vitality®, a semi-humid pet food for dogs that is commerciallyavailable, foodgrade binders, and a dried bacterial preparation of aEnterococcus faecium strain.

First, a premix was prepared of chicory flour (50% of premix), andpowdered FRISKIES Vitality® (25% of premix). This premix was dried in aconvection oven to a water activity close to zero (a_(w)≦0.01), andmoist maltodextrin (a_(w) about 0.3, 25% of premix) was mixed in tocomplete the premix.

Glycerol (3% of the weight of the premix) was sprayed on the surface ofthe premix powder to plasticize the surface of the powder particles.

By addition of the bacterial preparation, the mixture was completed.

The mixture was compacted to a cylindrical pellet (diameter: 1 cm,height about 1 cm) with a slightly convex top and bottom, with asingle-punch hydraulic laboratory press (Beckmann PT16). Compactionpressure was 3 ton/cm². The water activity of the pellet was 0.084 at25° C.

Half of the pellets were coated with a fat-based moisture barrier(Witocan 42/44 pastillen, Condea, France). Four coating layers wereapplied by dipping the pellets in a melt of the barrier material(temperature about 50° C.). The total amount of coating was about 15% ofthe uncoated pellets.

A schematic view of the coated pellets is given in FIG. 1.

EXAMPLE 2 Recovery of Micro-Organisms after Exposure to Humidity

The stability of coated pellets according to Example 1 were comparedwith the stability of micro-encapsulated E. faecium NCIMB 10415(commercialised as LBC-ME10) obtained from Cerbios-Pharma, Lugano,Switzerland, comprising about 5×10E+10 cfu/g. The micro-capsulescomprise the probiotic strain on a sucrose core that is then coated withseveral layers of undefined substances (food-grade moisture barriers)and the process to obtain these micro-capsules is largely unknown. Themicro-capsules are known to persist for a long time in semi-humidenvironment and are considered to be the best product currentlyavailable on the market.

Hence, the coated pellets according to Example 1 and micro-encapsulatedE. faecium NCIMB 10415 were exposed for 60 days to 30° C. and a humidenvironment (relative humidity of 70%). After different intervals,samples were taken and viable cell counts of E. faecium NCIMB 10415contained in the pellets and in the micro-capsules were determined

In FIG. 2 the recovery rate (in % of the initial cell count) of E.faecium SF68 in both samples is shown.

Remarkably, the compacted pellets performed better than the commerciallyavailable micro-capsules, especially after a storage time of 20 days.The recovery rate in the commercially available product decreasedstrongly and constantly, whereas the decrease in recovery ofmicro-organisms is prominently slower in the pellets according to theinvention.

EXAMPLE 3 Preparation of Different Pellets with Varying InnerMatrix-Components, Coatings and Micro-Organisms

Pellets according to the present invention were prepared by modifyingthe inner-matrix components, the coating and bacterial strains.

Bacterial Strains used in Pellets

1. Micro-encapsulated E. faecium NCIMB 10415 (commercialised asLBC-ME10). *

2. Lactobacillus johnsonii (CNCM-1225), freezedried, containing 15%amorphous carbohydrates.

3. Bifidobacterium lactis (DSM 20215), spraydried **

4. S. boulardii SB20, marketed as Levucell SB20**

* Obtained from Cerbios-Pharma, Lugano, Switzerland.

** Obtained from Christian Hansen BioSystems A/S (CHL), 10-12 Boge Allé,P.O Box 407, DK-2970 Horsholm, Denmark.

Inner Matrix Composition and Preparation of Pellets:

Matrix 1:

A. commercially available chicory flour (50 wt. %), B. Vitality® (25%,see Example 1), C. maltodextrin DE3 (25%) (Cerestar, France). ComponentsA and B are dried in an oven to a water activity <0.1. Glycerol (1-5 wt.%) is sprayed on using a spraying nozzle while the dry powder isagitated in a drum blender to ensure homogeneous dispersion of theglycerol. Component C is added at normal moisture level(0.25≦a_(w)<0.5). The bacterial preparation is added in (usually 0.1-5wt. % on total matrix, final dosage in pellet 10⁸ CFU/g). The mixture iscompacted at a pressure of 0.5 ton/cm², according to the process givenin Example 1.

Matrix 2:

Cerestar DC93000 direct compressible starch (Cerestar, France) Starch isdried to a_(w)<0.15. Glycerol (1-5 wt. %) is sprayed on using ahigh-pressure nozzle while the dry powder is agitated in a drum blender.Bacterial preparation is added in (usually 0.1-5 wt. % on total matrix,final dosage in pellet 10⁸ CFU/g). After addition of the bacterialculture, mixture is compacted at compaction pressures <0.5 ton/cm².

Matrix 3:

Lactose (50 wt. %) (Pharmatose DCL 15, DMV International, TheNetherlands), maltodextrin DE12 (50 wt. %) (Cerestar, France)Maltodextrin is dried to a_(w)<0.15, lactose is mixed in. Afterintroducing the bacterial preparation (usually 0.1-5 wt. % on totalmatrix, final dosage in pellet 10⁸ CFU/g) the mixture is compacted (evt.addition of 1-2 wt. % glycerol).

Coating of Pellets

Coating 1: Witocan 42/44 (see Example 1) (lipid-based coating)

Coating 2: Sepifilm LP010 (Seppic, France).

Coating is applied by fluidized-bed coating of the pellets using a GlattGPGC-3 coater. Sepifilm is applied as a 15% aqueous solution, totalamount of sepifilm on kibble mass=7-15%. Spraying pressure 1.5 bar,drying temperature 50° C., coating and drying time 45-90 min.

Results and Conclusion

Best recovery after storage time of about 30-60 days was obtained whenthe bacterial strains were present in particulate form. The use offragile bacterial cultures, for instance freeze-dried preparationswithout added carbohydrates, is less recommended when applied in kibblescompacted at high compaction pressures (>4 tons/cm²) as high losses inviability were observed. Granular preparations, for example containingsignificant amounts of carbohydrates in a spray dried bacterialpreparation, work very well.

In general, all inner matrixes worked similarly well, confirming thehigh variety possible for choosing components of the inner matrix.Regarding the moisture-barrier, best results were obtained with thefat-based moisture barrier, while the other types of moisture barrierprovide satisfactory results also.

The coating need to be applied properly, It is essential to guaranteethe quality and the integrity of the coating, as any cracks orstructural defects in the coatings will lead to rapid moisture uptake bythe kibbles and concomitantly to high losses in microbial viabilityduring storage.

1. A pellet comprising a compacted inner matrix and at least onecoating, wherein the inner matrix comprises viable micro-organisms andthe coating comprises a moisture barrier, characterised in that thepellet has a volume of at least 0.02 cm³.
 2. Pellet according to claim1, wherein the inner matrix, before or shortly after the coating, ischaracterised by a water activity of below 0.3.
 3. Pellet according toclaim 1, wherein the inner matrix has an envelope density of more than0.8 g/cm³.
 4. Pellet according to claim 1, which comprises 10⁵ to 10¹⁴viable micro-organisms.
 5. Pellet according to claim 1, wherein theinner matrix further comprises at least one component selected from thegroup of fillers, functional ingredients, lubricants, plasticizers,food-grade binders, and combinations thereof.
 6. Pellet according toclaim 1, wherein the inner matrix further comprises ingredients selectedfrom the group of digestible starches, resistant starches, other fibre,milled cereals, dried and milled vegetables, cellulose and cellulosederivatives, pet food, maltodextrin, chicory flour, protein isolates,yeast extracts, and mixtures thereof.
 7. Pellet according to claim 1,wherein the coating comprises a food-grade moisture barrier.
 8. Pelletswithin a food product, wherein the pellets are the pellets according toany of claim 1 to 7 and the inner matrix of the pellets comprises atleast one component of the food product.
 9. Delivery system ofprobiotics, which comprises the pellets according to any of claim 1 to8.
 10. Use of the pellets of any of claim 1 to 8 as a delivery systemfor probiotics in a moist, semi-moist, or semi-dry food product.
 11. Aprocess for obtaining a pellets to supplement a food product with viablemicro-organisms, which comprises the steps of mixing a preparation ofmicro-organisms and further components, drying the mixture to an a_(w)below 0.3, compacting the mixture under pressure to obtain pelletscomprising a volume of at least 0.02 cm³, and coating the pellets with amoisture barrier.
 12. The process according to claim 12, wherein thefurther components comprise at least part of the ingredients of the foodproduct.
 13. A food product comprising the pellets according to any ofclaims 1 to 7, wherein the food product and the inner matrix of thepellet share at least one component, ingredient or constituent. 14.Particulate food, food additive, supplement or pharmaceutical productconsisting of a mixture of at least two types of particles, one beingthe probiotic pellet according to any of claims 1 to 8.